Against the backdrop of global warming and exhaustion of fossil resources, production of chemical products using renewable resources, along with production of biofuels, is recognized as an emerging industry, biorefinery, which is an important means for realizing a low-carbon society, and has attracted attention.
4-Hydroxybenzoic acid is a useful substance as a raw material used to synthesize paraben, which is an antimicrobial agent, and also as a raw material for liquid-crystal polymers. Currently, 4-HBA is produced by chemical conversion from crude oil as a raw material. Examples of chemical 4-HBA production methods include a method in which phenol, potassium hydroxide, and carbon dioxide are reacted under high-pressure conditions. These methods depend on fossil materials for phenol as the starting material, and in addition, are energy-consumptive production processes requiring high-temperature and high-pressure conditions, which processes are typical in the chemical industry. Therefore, there is a need to establish an energy-saving, environment-conscious process that allows production of 4-HBA from renewable resources and produces less waste products, that is, to establish 4-HBA bioproduction technologies.
However, conventional bioproduction of 4-HBA from renewable resources is less productive as compared to bioproduction of lactic acid or ethanol because the metabolic reaction from a raw material sugar consists of a great many steps. In addition, there are problems, such as inhibition of bacterial growth by produced 4-HBA and cytotoxicity of 4-HBA. Therefore, industrial production of 4-HBA has not been achieved.
Using Escherichia coli, it has been revealed that 4-HBA is synthesized from chorismic acid, which is an intermediate in the shikimate pathway involved in the synthesis of aromatic amino acids etc., by chorismate-pyruvate lyase encoded by ubiC (Non Patent Literature 1 and 2, Patent Literature 1 and 2).
There is a report of introduction of a chorismate pyruvate-lyase gene (ubiC) of Escherichia coli into a different kind of microorganism, Klebsiella pneumoniae in an attempt to produce 4-HBA (Non Patent Literature 3). Also, there is a report of fermentative production of 4-HBA in an Escherichia coli in which the shikimic acid pathway is reinforced (Non Patent Literature 4). Furthermore, in an attempt to avoid the growth inhibition or the toxic action by 4-HBA, there are reports of selection of 4-HBA-resistant strains and of culture in the presence of an ion-exchange resin, but practically sufficient productivity of 4-HBA has not been achieved. Meanwhile, the inventors have released a report on the introduction of chorismate-pyruvate lyase into a coryneform bacterium and the production of phenol from glucose using the transformed coryneform bacterium. However, there is no description regarding the production of 4-HBA or the enzyme activity of chorismate-pyruvate lyase (Patent Literature 3).
Regarding other ubiC than that of Escherichia coli, the ubiC of Rhodobacter sphaeroides has been reported. However, both the transformant in which ubiC is highly expressed in Escherichia coli as a host and the transformant in which ubiC is highly expressed in Rhodobacter sphaeroides as a host are capable of producing 4-HBA only at low concentrations, and therefore, are not practically satisfactory (Patent Literature 4).
The UbiC of Escherichia coli has already been enzymatically analyzed in detail, and is known to be strongly inhibited by the product, 4-HBA. Therefore, in order to establish a high 4-HBA-producing strain that is industrially useful, obtaining a highly active ubiC, obtaining a resistant ubiC against product inhibition by 4-HBA, and selecting a 4-HBA resistant host are extremely important.